Immunohistochemical protocols for TGFβ1 (regulator of chondrocyte proliferation and differentiation27 (link)) and Indian hedgehog (Ihh; inhibits chondrocyte hypertrophy28 (link)) were performed on consecutive sections from 7-day (n=4) post-ischemia femoral heads and their contralateral controls (the same pigs as described for the hypertrophic cell and TRAP measurements). The TGFβ1 antibody was purchased from Invitrogen (#PA1–29032) and applied at a dilution of 1:100 following trypsin (Biocare) digestion. For Ihh, a polyclonal Ihh antibody from Novus Biologicals (NBP1–59443) was applied at a dilution of 1:100 following pepsin (Biocare) digestion. Both protocols utilized the Dako Rabbit Envision detection system with 3,3’-diaminobenzidine (DAB, brown staining) as chromogen and Harris hematoxylin as the counterstain. The distribution and immunoreactivity pattern (cytoplasmic, nuclear, membranous) were evaluated qualitatively for both TGFβ1 and Ihh.
Envision rabbit detection system
Manufactured by Agilent Technologies
The Envision rabbit detection system is a laboratory equipment product from Agilent Technologies. It is designed to provide a core function for detecting and analyzing samples. The description of its intended use or interpretation of its capabilities is not available.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin, by Biocare Medical (2 mentions)
Pa1 29032, by Biocare Medical (2 mentions)
Nbp1 59443, by Biocare Medical (2 mentions)
Pepsin, by Biocare Medical (2 mentions)
Ds ri2 camera, by Nikon (2 mentions)
Autostainer, by Agilent Technologies (2 mentions)
Automated slide stainer, by Agilent Technologies (1 mentions)
Mayer s hematoxylin, by Merck Group (1 mentions)
Pt link module, by Agilent Technologies (1 mentions)
Mayer s hematoxylin, by Agilent Technologies (1 mentions)
Elements d version 4, by Nikon (1 mentions)
5 protocols using envision rabbit detection system
1
Immunohistochemical Analysis of TGFβ1 and Ihh
2
Assessing TGFβ1 and Ihh in Femoral Head Ischemia
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Immunohistochemical protocols for TGFβ1 (regulator of chondrocyte proliferation and differentiation 27 ) and Indian hedgehog (Ihh; inhibits chondrocyte hypertrophy 28 ) were performed on consecutive sections from 7-day (n=4) post-ischemia femoral heads and their contralateral controls (the same pigs as described for the hypertrophic cell and TRAP measurements). The TGFβ1 antibody was purchased from Invitrogen (#PA1-29032) and applied at a dilution of 1:100 following trypsin (Biocare) digestion. For Ihh, a polyclonal Ihh antibody from Novus Biologicals (NBP1-59443) was applied at a dilution of 1:100 following pepsin (Biocare) digestion. Both protocols utilized the Dako Rabbit Envision detection system with 3,3'-diaminobenzidine (DAB, brown staining) as chromogen and Harris hematoxylin as the counterstain. The distribution and immunoreactivity pattern (cytoplasmic, nuclear, membranous) were evaluated qualitatively for both TGFβ1 and Ihh.
3
Immunohistochemical Detection of TOX3 in Breast Cancer
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Immunohistochemical detection of TOX3 was performed on 4-μm sections of formalin fixed paraffin embedded tissue stained with AJ-33. Staining was done using an automated slide stainer (Dako, Carpinteria, California, USA). Antigen retrieval was performed using the Dako PT Link Module and low pH buffer. Staining was visualized using the Dako Envision + Rabbit Detection System. Slides were subsequently counterstained with Mayer’s hematoxylin (Sigma-Aldrich). Three commercially available tissue arrays (Pantomics, Richmond, CA, USA) that contained a total of 210 primary breast cancer samples in duplicate were analyzed for TOX3 expression. Some samples were destroyed during the processing and therefore removed from analysis. Thus, the TOX3 histological data shown here are derived from a total of 188 breast tumors. Other histological data that were associated with individual tumors on the tissue array were supplied by the manufacturer. For some histological analysis, the Department of Pathology and Laboratory Medicine at CSMC supplied normal breast tissue obtained following mammoplasty. Samples were obtained under a waiver of consent and provided in an anonymous fashion, so that the connection to individual patients was destroyed prior to their analysis. This work was performed under Cedars-Sinai Medical Center’s Institutional Review Board Study Number: Pro 00033387.
4
Immunohistochemical Analysis of SRGAP2 in Bone Tissue
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Tissue microarrays were purchased from Biomax (Normal bone: BO244d, Osteosarcoma: OS804a). Slides containing 4 μm thick formalin-fixed, paraffin-embedded sections of tumor/control bone tissue were deparaffinized and rehydrated. Antigen retrieval was performed in a steamer using 1 mM Tris base EDTA buffer, pH 9.0. After endogenous peroxidase blocking, a protein block was applied. Immunohistochemistry (IHC) for SRGAP2 was performed using rabbit anti-SRGAP2 (Sigma-Aldrich HPA-028191) primary antibody on a Dako Autostainer. Detection was achieved using the Dako Envision rabbit detection system with diaminobenzidine (Dako, Glostrup) as the chromogen. Sections were counterstained with Mayer’s Hematoxylin (Dako). Xenograft tumors confirmed by western blot to express SRGAP2 were used as positive control.
Tissue sections were imaged on a Nikon E800M microscope at 40X magnification using a Nikon DSRi2 camera and Nikon Elements D Version 4 software. Each section was imaged using the same white balance and shading correction settings. Whole image sharpness was uniformly adjusted with Photoshop Elements version 11 software.
Tissue sections were imaged on a Nikon E800M microscope at 40X magnification using a Nikon DSRi2 camera and Nikon Elements D Version 4 software. Each section was imaged using the same white balance and shading correction settings. Whole image sharpness was uniformly adjusted with Photoshop Elements version 11 software.
5
Immunohistochemical Analysis of SEMA4C in Osteosarcoma
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The osteosarcoma tissue microarray was purchased from Biomax (Osteosarcoma: OS804c) containing 40 samples in duplicate. Slides containing 4 μm thick formalin-fixed, paraffin-embedded sections of tumor tissue were deparaffinized and rehydrated. Antigen retrieval was performed in a steamer using 1 mM Tris base EDTA buffer, pH 9.0. After endogenous peroxidase blocking, a protein block was applied. Immunohistochemistry (IHC) for SEMA4C was performed using a rabbit anti-SEMA4C primary antibody (#AF6125, R & D Systems) on an autostainer (Dako). Detection was achieved using the Envision rabbit detection system (Dako) with diaminobenzidine (DAB) as the chromogen. Tissue sections were imaged on a Nikon E800M microscope at 40X magnification using a Nikon DSRi2 camera and Nikon Elements D Version 4 software. Slides were evaluated and scored as previously described [19] .
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